A variety of service and collaborative projects in protein characterization have been or are being carried out within the Mass Spectrometry Research and Support Group with approximately 4000 samples analyzed from 39 scientists representing 23 principal investigators or core heads from 6 laboratory branches and the DNTP. One large effort is in support of the Protein Expression Core Facility (PECF) and Dr. Bob Petrovich. The Role of the PMCF is to confirm gene expression at the protein level prior to the PECF handing materials over to their users. Other published and unpublished projects that are still ongoing include: Characterization of dust and proteins from allergens in collaboration - Don Cook, Geoffrey Mueller, and Robert London Characterization of protein:protein cross-links in RNA processing enzymes Robin Stanley Characterization of post-translational modifications on the CRM1 protein Raja Jothi Characterization of phosphorylation sites on the serine/threonine kinase VRK1 Masahiko Negishi Characterization of adenylation on DNA Ligase IV Andrea Moon and Lars Pedersen Identification of bacterial pathogens by MALDI-MS David Kurtz Other projects that have been recently published, or have been submitted and accepted for publication include: With Dr. R.Scott Williams: Topoisomerase 2 (TOP2) DNA transactions are essential for life, and proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein crosslinks are resolved is unclear. Here, we show that the SUMO ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent Tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a split-SIM SUMO2 engagement platform. These findings uncover a ZATTTDP2 catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc. The specific contribution of the Mass Spectrometry Research and Support Group was to identify the proteins that immunoprecipitated with Tdp2. This resulted in the discovery of ZATT as a factor involved in DNA repair. With Dr. Robin Stanley: Las1 is a recently discovered endoribonuclease that collaborates with Grc3-Rat1-Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. We reported that the essential Las1 endoribonuclease requires it binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results established that Grc3 specifically directs Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively towards single-stranded RNA. Together Las1 and Grc3 assemble into a tetrameric complex that is required for competenent rRNA processing. The tetrameric Grc3/Las1 crosstalk draws unexpected parallels to endoribonucleases RNaseL and Ire1 and establishes Grc3/Las1 as a novel member of the RNaseL/Ire1 RNA Splicing Family. Together, our work provided mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a new example of a protein guided programmable endoribonuclease. The specific contribution of the Mass Spectrometry Research and Support Group was to characterize the recombinant proteins used in these experiments as well as to identify the cleavage site in the RNA substrate. Additional projects that have required more than negligible resources include efforts performed with the Blackshear, Cidlowski, Garantziotis, Hall, Hu, Kunkel, and Wilson laboratories.